Design, Synthesis, and Biological Evaluation of 5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines as Microtubule Targeting Agents

A series of eleven 4-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines were designed and synthesized and their biological activities were evaluated. Synthesis involved the Gewald reaction to synthesize ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate ring, and SNAr reactions. Compound 4 was 1.6- and ~7-fold more potent than the lead compound 1 in cell proliferation and microtubule depolymerization assays, respectively. Compounds 4, 5 and 7 showed the most potent antiproliferative effects (IC50 values < 40 nM), while compounds 6, 8, 10, 12 and 13 had lower antiproliferative potencies (IC50 values of 53–125 nM). Additionally, compounds 4–8, 10 and 12–13 circumvented Pgp and βIII-tubulin mediated drug resistance, mechanisms that diminish the clinical efficacy of paclitaxel (PTX). In the NCI-60 cell line panel, compound 4 exhibited an average GI50 of ~10 nM in the 40 most sensitive cell lines. Compound 4 demonstrated statistically significant antitumor effects in a murine MDA-MB-435 xenograft model.


Introduction
Microtubules have long been recognized as effective targets for the treatment of many human malignancies [1,2]. Microtubules are involved in a variety of cellular functions including mitosis, motility, intracellular transport, trafficking and organization, including positioning of organelles [1,3,4]. Molecules binding to tubulin and interrupting tubulin dynamics are recognized as microtubule targeting agents (MTAs), and they have been used clinically as single agents or in combinatorial regimens for the effective treatment of leukemia, lymphoma and various solid tumors [2,3,5]. MTAs are a highly diverse class of cytotoxic agents that include a variety of different chemical scaffolds ( Figure 1) [2,3,6].
MTAs can be classified into two major groups: (1) microtubule destabilizers that initiate microtubule depolymerization; and (2) microtubule stabilizers that promote the polymerization of tubulin into microtubules [1,2]. Additionally, MTAs are further divided into seven groups based on their binding sites [6,7]. Two binding sites on tubulin/microtubules have been identified for microtubule stabilizers [8]. First, the taxane site is located on the interior of the microtubule, and all clinically approved microtubule stabilizers, including paclitaxel ( Figure 1), docetaxel (Figure 1), cabazitaxel and ixabepilone, bind to this site [8]. MTAs can be classified into two major groups: (1) microtubule destabilizers that initiate microtubule depolymerization; and (2) microtubule stabilizers that promote the polymerization of tubulin into microtubules [1,2]. Additionally, MTAs are further divided into seven groups based on their binding sites [6,7]. Two binding sites on tubulin/microtubules have been identified for microtubule stabilizers [8]. First, the taxane site is located on the interior of the microtubule, and all clinically approved microtubule stabilizers, including paclitaxel (Figure 1), docetaxel (Figure 1), cabazitaxel and ixabepilone, bind to this site [8]. The taccalonolides (Figure 1), zampanolide and cyclostreptin are compounds that bind covalently within the taxane site, but to date have not been evaluated clinically [8][9][10]. The second stabilizer site is the laulimalide/peloruside site, which is located on the exterior of the microtubule and named for the natural products that bind to this site [8].
The clinical development of compounds binding to this site has been limited by a lack of in vivo efficacy for laulimalide [11] and supply challenges for peloruside A [12]. In the class of microtubule destabilizers, five sites have been identified: the vinca site, the colchicine site (CS), the maytansine site, the pironetin site [6], and more recently the gatorbulin site (gatorbulin-1, Figure 1) defined by the cyclic peptide of the same name [7]. The vinca alkaloids vinblastine, vincristine, vindesine, and vinorelbine, as well as other structurally unique/unrelated compounds, including eribulin (Figure 1), bind within the vinca site, which is located at the interdimer interface between two tubulin heterodimers in a protofilament [6]. The colchicine binding site is located on β-tubulin at the intradimer interface While the clinically useful vinca alkaloids vinblastine and vincristine were approved decades ago, new MTAs have been approved more recently for clinical use. Eribulin (Figure 1), a simplified synthetic analogue of the natural product halichondrin B, is a microtubule depolymerizer that has unique properties [5,15] and significant utility in the treatment of advanced breast cancer [15]. The dolastatin 10 analogue monomethyl auristatin E and maytansine ( Figure 1) analogues are employed as the cytotoxic payloads of antibody-drug conjugates (ADCs) that have found clinical utility [16]. These unconjugated MTAs were too toxic for systemic administration, but their antibody-directed delivery to cancers was designed to reduce off-target toxicities [16]. Continuing challenges with clinically approved MTAs, including the taxanes and vinca alkaloids, are the incidence of dose-limiting side effects and limited efficacy due to multidrug resistance [17]. Cancer cells and patients demonstrate resistance to clinically used agents as a result of the expression of the drug efflux pump P-glycoprotein (Pgp) and the βIII-tubulin isotype [2,18,19], leading to efforts to identify new MTAs that can overcome these mechanisms of drug resistance.  Figure 1), have been extensively studied, and several have been evaluated in clinical trials, including combretastatin A-4 phosphate (CA-4P/fosbretabulin), the combretastatin CA-1P prodrug (OXi4503), 2-methoxyestradiol, AVE8062, CKD-516, BNC105P, ABT-751, CYT-997, ZD6126, plinabulin (NPI-2358) and MN-029 [20][21][22]. Colchicine itself is approved for the treatment of gout but is not employed as an anticancer agent due to toxic side effects at the doses necessary for efficacy [23]. However, other CS agents have exhibited promising potential as anticancer candidates [20][21][22]. Many compounds that interact with the CS are able to overcome multiple mechanisms of drug resistance [24]. This suggests that the development of MTAs targeting the CS has the potential to overcome limitations associated with existing drugs and perhaps improve clinical outcomes. This has been challenging, however, and to date no CS agent has received FDA approval for anticancer indications [22]. There is an urgent need to develop new tubulin inhibitors with fewer side effects and good oral bioavailability that are less prone to clinically relevant drug resistance mechanisms.

Rationale
We previously reported [25,26] N4-substituted-pyrimido [4,5- (1) Isosteric replacement: To explore the activities of compounds with the 4,5,6,7tetrahydrobenzo thiophene scaffold on both inhibition of cancer cell proliferation and microtubule depolymerization, we carried out the isosteric replacement of the scaffold -NH-of the lead compounds 1-3 by sulfur (-S-) to afford target compounds 4-14 (Table 1). Isosteric replacement of -NH with (-S-) has literature precedence in improving antiproliferative and microtubule depolymerizing activities [27]. Moreover, pharmacological applications of 5,6,7,8-tetrahydrobenzo [4,5]thieno [2,3-d]pyrimidines have been extensively illustrated in various reports in the literature [28][29][30][31][32][33][34][35][36][37][38]. In addition, the lead tricyclic compounds and the proposed target compounds incorporate a p-methoxyphenyl substitution akin to colchicine and CA-4 ( Figure 1). The nature of the heteroatom substitution (S for NH) affects hydrogen bond (HB) strength [39]. Thus, it was also of interest to isosterically replace the oxygen atom of the 4 -OCH 3 of 4, 8 and 9 with a sulfur moiety to afford 5, 10, and 11, in analogy to 2. (2) Decrease numbers of sp2 bonds: Drug candidates show a higher clinical success rate with one or more sp3 hybridized carbon atoms as compared to "flat" molecules, due to low aqueous solubility of purely aromatic compounds [40]. One of the major limitation of some MTAs, particularly the taxanes, is their poor water solubility [41]. Thus, watersoluble MTAs are highly coveted, and an enormous effort continues to chemically modify and/or formulate analogues to increase their water solubility. Increasing 'aromatic proportion' in a molecule has a detrimental effect on the solubility [40]. The fraction of sp3 hybridized carbon atoms (Fsp3), in other words, the fraction of carbon atoms that are saturated, correlates positively with water solubility [40]. In an attempt to both increase the water solubility as well to probe the potential interactions with the hydrophobic pocket in the CS, we designed target compounds 4-14 by incorporating sp3 hybridized carbon atoms in the tricyclic scaffold of the lead compounds 1-3. 2-NH 2 on compound 4 with a 2-CH 3 to afford 8. This would also provide information regarding the activity on the replacement of H with CH 3 at the 2-position in the tricyclic scaffold. (4) Conformational restriction: Conformational restriction or rigidification of a ligand can decrease the entropic penalty [42]. The ligand can adopt a preferred conformation for binding, which might lead to enhanced potency for a given physiological target [42].
In an effort to better define the conformational requirements for biological activities, we systematically incorporated various groups to restrict bond rotations. The conformation of 9 ( Figure 3) is determined by three rotatable single bonds: the 4-position C-N bond (bond a), the 1 -position C-N bond (bond b) and the 4 -position C-O bond (bond c). Conformational analysis via molecular modeling and 1 H NMR studies [25] suggest that the methyl group on the aniline nitrogen in 1 restricted the free rotation of bond a as well as bond b ( Figure 2) and consequently restricted the conformation of the anilino ring. To study the significance of conformational restriction on biological activities, we first designed compounds 8 and 9. In 9, the rotation of bonds a and b was restricted by incorporating a methyl group at the N4-position to afford compound 8.
Incorporation of tetrahydroquinoline rings in 6 and 12 further restricted bond b of 4 and 8. The design of compound 13 via the incorporation of a 5-methoxy naphthalene ring provided a further element of conformational restriction.
by incorporating sp3 hybridized carbon atoms in the tricyclic scaffold of the lead compounds 1-3. (3) Variation of the substituents at the 2-position: Compound 7 was specifically designed to determine the effect of replacing the 2-NH2 in 4 with a 2-H. This allows an exploration of the 2-NH2 and hydrogen bond interactions with corresponding amino acids at the CS. It was also of our interest to observe the effect of isosteric replacement of 2-NH2 on compound 4 with a 2-CH3 to afford 8. This would also provide information regarding the activity on the replacement of H with CH3 at the 2-position in the tricyclic scaffold. (4) Conformational restriction: Conformational restriction or rigidification of a ligand can decrease the entropic penalty [42]. The ligand can adopt a preferred conformation for binding, which might lead to enhanced potency for a given physiological target [42]. In an effort to better define the conformational requirements for biological activities, we systematically incorporated various groups to restrict bond rotations. The conformation of 9 ( Figure 3) is determined by three rotatable single bonds: the 4position C-N bond (bond a), the 1′-position C-N bond (bond b) and the 4′-position C-O bond (bond c). Conformational analysis via molecular modeling and 1 H NMR studies [25] suggest that the methyl group on the aniline nitrogen in 1 restricted the free rotation of bond a as well as bond b ( Figure 2) and consequently restricted the conformation of the anilino ring. To study the significance of conformational restriction on biological activities, we first designed compounds 8 and 9. In 9, the rotation of bonds a and b was restricted by incorporating a methyl group at the N4-position to afford compound 8. Incorporation of tetrahydroquinoline rings in 6 and 12 further restricted bond b of 4 and 8. The design of compound 13 via the incorporation of a 5-methoxy naphthalene ring provided a further element of conformational restriction.     by incorporating sp3 hybridized carbon atoms in the tricyclic scaffold of the lead compounds 1-3. (3) Variation of the substituents at the 2-position: Compound 7 was specifically designed to determine the effect of replacing the 2-NH2 in 4 with a 2-H. This allows an exploration of the 2-NH2 and hydrogen bond interactions with corresponding amino acids at the CS. It was also of our interest to observe the effect of isosteric replacement of 2-NH2 on compound 4 with a 2-CH3 to afford 8. This would also provide information regarding the activity on the replacement of H with CH3 at the 2-position in the tricyclic scaffold. (4) Conformational restriction: Conformational restriction or rigidification of a ligand can decrease the entropic penalty [42]. The ligand can adopt a preferred conformation for binding, which might lead to enhanced potency for a given physiological target [42]. In an effort to better define the conformational requirements for biological activities, we systematically incorporated various groups to restrict bond rotations. The conformation of 9 ( Figure 3) is determined by three rotatable single bonds: the 4position C-N bond (bond a), the 1′-position C-N bond (bond b) and the 4′-position C-O bond (bond c). Conformational analysis via molecular modeling and 1 H NMR studies [25] suggest that the methyl group on the aniline nitrogen in 1 restricted the free rotation of bond a as well as bond b ( Figure 2) and consequently restricted the conformation of the anilino ring. To study the significance of conformational restriction on biological activities, we first designed compounds 8 and 9. In 9, the rotation of bonds a and b was restricted by incorporating a methyl group at the N4-position to afford compound 8. Incorporation of tetrahydroquinoline rings in 6 and 12 further restricted bond b of 4 and 8. The design of compound 13 via the incorporation of a 5-methoxy naphthalene ring provided a further element of conformational restriction.   by incorporating sp3 hybridized carbon atoms in the tricyclic scaffold of the lead compounds 1-3. (3) Variation of the substituents at the 2-position: Compound 7 was specifically designed to determine the effect of replacing the 2-NH2 in 4 with a 2-H. This allows an exploration of the 2-NH2 and hydrogen bond interactions with corresponding amino acids at the CS. It was also of our interest to observe the effect of isosteric replacement of 2-NH2 on compound 4 with a 2-CH3 to afford 8. This would also provide information regarding the activity on the replacement of H with CH3 at the 2-position in the tricyclic scaffold. (4) Conformational restriction: Conformational restriction or rigidification of a ligand can decrease the entropic penalty [42]. The ligand can adopt a preferred conformation for binding, which might lead to enhanced potency for a given physiological target [42]. In an effort to better define the conformational requirements for biological activities, we systematically incorporated various groups to restrict bond rotations. The conformation of 9 ( Figure 3) is determined by three rotatable single bonds: the 4position C-N bond (bond a), the 1′-position C-N bond (bond b) and the 4′-position C-O bond (bond c). Conformational analysis via molecular modeling and 1 H NMR studies [25] suggest that the methyl group on the aniline nitrogen in 1 restricted the free rotation of bond a as well as bond b ( Figure 2) and consequently restricted the conformation of the anilino ring. To study the significance of conformational restriction on biological activities, we first designed compounds 8 and 9. In 9, the rotation of bonds a and b was restricted by incorporating a methyl group at the N4-position to afford compound 8. Incorporation of tetrahydroquinoline rings in 6 and 12 further restricted bond b of 4 and 8. The design of compound 13 via the incorporation of a 5-methoxy naphthalene ring provided a further element of conformational restriction.   by incorporating sp3 hybridized carbon atoms in the tricyclic scaffold of the lead compounds 1-3. (3) Variation of the substituents at the 2-position: Compound 7 was specifically designed to determine the effect of replacing the 2-NH2 in 4 with a 2-H. This allows an exploration of the 2-NH2 and hydrogen bond interactions with corresponding amino acids at the CS. It was also of our interest to observe the effect of isosteric replacement of 2-NH2 on compound 4 with a 2-CH3 to afford 8. This would also provide information regarding the activity on the replacement of H with CH3 at the 2-position in the tricyclic scaffold. (4) Conformational restriction: Conformational restriction or rigidification of a ligand can decrease the entropic penalty [42]. The ligand can adopt a preferred conformation for binding, which might lead to enhanced potency for a given physiological target [42]. In an effort to better define the conformational requirements for biological activities, we systematically incorporated various groups to restrict bond rotations. The conformation of 9 ( Figure 3) is determined by three rotatable single bonds: the 4position C-N bond (bond a), the 1′-position C-N bond (bond b) and the 4′-position C-O bond (bond c). Conformational analysis via molecular modeling and 1 H NMR studies [25] suggest that the methyl group on the aniline nitrogen in 1 restricted the free rotation of bond a as well as bond b ( Figure 2) and consequently restricted the conformation of the anilino ring. To study the significance of conformational restriction on biological activities, we first designed compounds 8 and 9. In 9, the rotation of bonds a and b was restricted by incorporating a methyl group at the N4-position to afford compound 8. Incorporation of tetrahydroquinoline rings in 6 and 12 further restricted bond b of 4 and 8. The design of compound 13 via the incorporation of a 5-methoxy naphthalene ring provided a further element of conformational restriction.

IC50 ± SD in MDA-435 Cells (nM)
EC50 for Microtubule Depolymerization in A-10 Cells (nM) 1 14.7 ± 1.5 130 2 89.1 ± 10 1100 3 130 ± 7.8 1200  by incorporating sp3 hybridized carbon atoms in the tricyclic scaffold of the lead compounds 1-3. (3) Variation of the substituents at the 2-position: Compound 7 was specifically designed to determine the effect of replacing the 2-NH2 in 4 with a 2-H. This allows an exploration of the 2-NH2 and hydrogen bond interactions with corresponding amino acids at the CS. It was also of our interest to observe the effect of isosteric replacement of 2-NH2 on compound 4 with a 2-CH3 to afford 8. This would also provide information regarding the activity on the replacement of H with CH3 at the 2-position in the tricyclic scaffold. (4) Conformational restriction: Conformational restriction or rigidification of a ligand can decrease the entropic penalty [42]. The ligand can adopt a preferred conformation for binding, which might lead to enhanced potency for a given physiological target [42]. In an effort to better define the conformational requirements for biological activities, we systematically incorporated various groups to restrict bond rotations. The conformation of 9 ( Figure 3) is determined by three rotatable single bonds: the 4position C-N bond (bond a), the 1′-position C-N bond (bond b) and the 4′-position C-O bond (bond c). Conformational analysis via molecular modeling and 1 H NMR studies [25] suggest that the methyl group on the aniline nitrogen in 1 restricted the free rotation of bond a as well as bond b ( Figure 2) and consequently restricted the conformation of the anilino ring. To study the significance of conformational restriction on biological activities, we first designed compounds 8 and 9. In 9, the rotation of bonds a and b was restricted by incorporating a methyl group at the N4-position to afford compound 8. Incorporation of tetrahydroquinoline rings in 6 and 12 further restricted bond b of 4 and 8. The design of compound 13 via the incorporation of a 5-methoxy naphthalene ring provided a further element of conformational restriction.

EC50 for Microtubule Depolymerization in A-10
89.1 ± 10 1100 3 130 ± 7.8 1200  by incorporating sp3 hybridized carbon atoms in the tricyclic scaffold of the lead compounds 1-3. (3) Variation of the substituents at the 2-position: Compound 7 was specifically designed to determine the effect of replacing the 2-NH2 in 4 with a 2-H. This allows an exploration of the 2-NH2 and hydrogen bond interactions with corresponding amino acids at the CS. It was also of our interest to observe the effect of isosteric replacement of 2-NH2 on compound 4 with a 2-CH3 to afford 8. This would also provide information regarding the activity on the replacement of H with CH3 at the 2-position in the tricyclic scaffold. (4) Conformational restriction: Conformational restriction or rigidification of a ligand can decrease the entropic penalty [42]. The ligand can adopt a preferred conformation for binding, which might lead to enhanced potency for a given physiological target [42]. In an effort to better define the conformational requirements for biological activities, we systematically incorporated various groups to restrict bond rotations. The conformation of 9 ( Figure 3) is determined by three rotatable single bonds: the 4position C-N bond (bond a), the 1′-position C-N bond (bond b) and the 4′-position C-O bond (bond c). Conformational analysis via molecular modeling and 1 H NMR studies [25] suggest that the methyl group on the aniline nitrogen in 1 restricted the free rotation of bond a as well as bond b ( Figure 2) and consequently restricted the conformation of the anilino ring. To study the significance of conformational restriction on biological activities, we first designed compounds 8 and 9. In 9, the rotation of bonds a and b was restricted by incorporating a methyl group at the N4-position to afford compound 8. Incorporation of tetrahydroquinoline rings in 6 and 12 further restricted bond b of 4 and 8. The design of compound 13 via the incorporation of a 5-methoxy naphthalene ring provided a further element of conformational restriction.

Molecular Modeling
Computational modeling studies were performed to elucidate the binding mode of the lead and target compounds 1-14 and probe the possible interactions with the CS. Compounds 1-14 were docked into the CS (PDB: 6BS2, 2.65 Å) [43] of tubulin using Maestro, Schrödinger 2020-2, New York, NY, USA [44]. Figure 4a shows the docked pose of 4 (cyan) superimposed with colchicine (pink) in the X-ray crystal structure of the CS [43].

Molecular Modeling
Computational modeling studies were performed to elucidate the binding mode of the lead and target compounds 1-14 and probe the possible interactions with the CS. Compounds 1-14 were docked into the CS (PDB: 6BS2, 2.65 Å) [43] of tubulin using Maestro, Schrödinger 2020-2, New York, NY, USA [44]. Figure 4a shows the docked pose of 4 (cyan) superimposed with colchicine (pink) in the X-ray crystal structure of the CS [43].

Molecular Modeling
Computational modeling studies were performed to elucidate the binding mode of the lead and target compounds 1-14 and probe the possible interactions with the CS. Compounds 1-14 were docked into the CS (PDB: 6BS2, 2.65 Å) [43] of tubulin using Maestro, Schrödinger 2020-2, New York, NY, USA [44]. Figure 4a shows the docked pose of 4 (cyan) superimposed with colchicine (pink) in the X-ray crystal structure of the CS [43].

Molecular Modeling
Computational modeling studies were performed to elucidate the binding mode of the lead and target compounds 1-14 and probe the possible interactions with the CS. Compounds 1-14 were docked into the CS (PDB: 6BS2, 2.65 Å) [43] of tubulin using Maestro, Schrödinger 2020-2, New York, NY, USA [44]. Figure 4a shows the docked pose of 4 (cyan) superimposed with colchicine (pink) in the X-ray crystal structure of the CS [43].  Table S1). The pyrimidine ring of 4 overlaps with the pyrimidine ring of the crystalized ligand of 6BS2 (Supplementary Materials Figure S1).  Table  S1). The pyrimidine ring of 4 overlaps with the pyrimidine ring of the crystalized ligand of 6BS2 (Supplementary Materials Figure S1).

Chemistry
Compounds 4-14 were synthesized according to the synthetic routes outlined in Schemes 1-4. The Gewald reaction (Scheme 1) was carried out on a solution of sulfur in ethanol, to which cyclohexanone 15 and ethyl cyanoacetate were added. Morpholine was added dropwise to the solution to obtain 16. Cyclization of 16 with chloro-formamidine hydrochloride, formamide and acetonitrile afforded 17, 18 and 19, respectively, using reported methods [25,45,46]. Chlorination [47]

Chemistry
Compounds 4-14 were synthesized according to the synthetic routes outlined in Schemes 1-4. The Gewald reaction (Scheme 1) was carried out on a solution of sulfur in ethanol, to which cyclohexanone 15 and ethyl cyanoacetate were added. Morpholine was added dropwise to the solution to obtain 16. Cyclization of 16 with chloro-formamidine hydrochloride, formamide and acetonitrile afforded 17, 18 and 19, respectively, using reported methods [25,45,46]. Chlorination [47] Table  S1). The pyrimidine ring of 4 overlaps with the pyrimidine ring of the crystalized ligand of 6BS2 (Supplementary Materials Figure S1).

Antiproliferative and Microtubule Depolymerization Effects
We investigated the microtubule depolymerization and the antiproliferative activities of compounds 4-14 (Table 2). At a concentration of 10 µM, the compounds that caused at least 50% microtubule depolymerization were further evaluated to determine their EC 50 values, the concentration that causes the loss of 50% of cellular microtubules as visualized microscopically. Compounds that caused microtubule depolymerization at 10 µM were further evaluated for antiproliferative potency in the drug-sensitive MDA-MB-435 cancer cell line, and the IC 50 (concentration required to cause 50% inhibition of proliferation) values were determined using the sulforhodamine B assay (SRB assay). Compound 4, the 2-NH 2 analogue of 5,6,7,8-tetrahydrobenzo [4,5]thieno [2,3-d]pyrimidine, was the most potent compound of this series for both microtubule depolymerizing and antiproliferative effects, with an EC 50 of 19 nM and an IC 50 of 9.0 nM ( Table 2). Compound 4 was 7-fold more potent than 1 for microtubule depolymerizing effects, indicating that the 5,6,7,8tetrahydrobenzo [4,5]thieno [2,3-d]pyrimidine ring is significantly better for microtubule depolymerizing activity than the pyrimido [4,5-b]indole ring of 1 ( Table 2) and that it additionally contributes to improvements in antiproliferative potency. We next evaluated the importance of the 4 -OMe group of compound 4 by replacing it with an isosteric 4 -SMe (5). The resulting compound 5 was 15-fold more potent than the corresponding lead compound 2 with respect to microtubule depolymerization activity and was additionally 2.3-fold more potent for antiproliferative effects compared to 2. Clearly, this further substantiated the importance of an S in the scaffold over an NH. However, comparing compounds 4 and 5 indicated that the 4 -OMe was better than the 4 -SMe. Compound 6, the tetrahydroquinoline-substituted compound, a conformationally restricted analogue of 4 around bond b, was 6-fold less potent for antiproliferative effects and for microtubule depolymerizing effects than 4, indicating that conformational restriction in 6 is detrimental to these biological activities. We next focused on substituting the 2-position of the 5,6,7,8-tetrahydrobenzo [4,5] thieno [2,3-d]pyrimidine. The corresponding 2-H analogue 7 of lead compound 3 displayed 27-fold increased potency in the microtubule depolymerizing assay as compared with 3 (Table 2), indicating a more effective engagement with tubulin. Compound 7 was 3.5-fold more potent than compound 3 for antiproliferative effects. The 2-Me analogue 8 displayed slightly less potency compared to the 2-H compound 7 for both microtubule depolymerizing and antiproliferative effects, yet it had 2.7-and 5.8-fold lower potency than the 2-NH 2 compound 4 in the microtubule depolymerization assay and antiproliferative assay, re-spectively. Compound 8 however, with a 2-Me, had a lower EC 50 /IC 50 ratio (1.0 for 8 as compared to 2.2 for 4), indicating a tighter correlation between the microtubule depolymerizing effects and the cancer cell cytotoxicity. Compound 10 with a 4 -SMe group and a 2-Me analogue of compound 5 had 2-fold lower potency than 5 in both assays.
In compounds 12 and 13, conformational restrictions of the N4-phenyl moiety of 8 about bond b with a 1,2,3,4-tetrahydroquinoline moiety and 5 -methoxy naphthalene, respectively, caused a 2.3-3-fold decrease in potency for 12 in antiproliferative and microtubule depolymerizing effects compared to 8 and a 1.5-to 2-fold drop in potency for 13 as compared to 8. Compounds 9, 11, and 14 did not show any activity in the microtubule depolymerization assay, and these were not evaluated for antiproliferative effects, corroborating our previous reports that the N4-Me is crucial for MT activity [26].

Inhibition of Tubulin Assembly and Colchicine Binding
Compounds 4, 5, 7, 8 and 10 were evaluated for their direct effects on purified tubulin assembly and for inhibition of colchicine binding (Table 3). Compounds 4, 5, 7, 8 and 10 inhibited tubulin assembly with activities better than those of the lead compounds 1-3 as well as CA-4. Compound 4 was 2-fold more potent than the lead 1 as an inhibitor of tubulin assembly. On the other hand, compounds 5, 7, 8 and 10 were 2-fold more potent than the standard CA-4. Moreover, compounds 5 and 7 were 5-fold more potent as inhibitors of tubulin assembly than the corresponding lead compounds 2 and 3, respectively. Compounds 4, 5, 7, 8 and 10 inhibited the binding of [ 3 H]colchicine to tubulin by 89-99%, whereas the lead compounds 1, 2 and 3 showed 84, 67, and 62% inhibition of [ 3 H]colchicine binding, respectively. Thus 4, 5, 7, 8 and 10 were more active than the initial lead compounds 1-3. These results clearly demonstrated that these compounds are CS MTAs. Table 3. Inhibition of tubulin assembly and colchicine binding.

Compound
Inhibition of Tubulin Assembly IC 50

Effect on βIII-Tubulin and Pgp-Mediated Cancer Cell Resistance
Compounds 4-8, 10, 12, and 13 were evaluated for their abilities to overcome βIIItubulin mediated drug resistance using an isogenic HeLa cell line pair (Table 4). Consistent with the results obtained in MDA-MB-535 cells, compound 4 was the most potent in the series in the HeLa and HeLa WT βIII cell lines, with 1.6-fold higher potency than the lead compound 1. Compounds 5 and 7 showed 2-fold higher potency than the lead compounds 2 and 3 in HeLa and HeLa WT βIII cell lines. The Rr values (Table 4) were calculated by dividing the IC 50 of the βIII-tubulin expressing line by the IC 50 obtained in the parental HeLa cells. The expression of βIII-tubulin is known to lead to paclitaxel resistance, and paclitaxel has an Rr value of 8.6 in this cell line pair (Table 4). The target compounds 3-8, 10, 12, and 13 have Rr values~1.0 ( Table 4), suggesting that they circumvent βIII-tubulin mediated drug resistance, in contrast to paclitaxel. The potent MTAs 4-8, 10, 12, and 13 were also evaluated for their activity in the SK-OV-3 ovarian carcinoma cell line and the Pgp-expressing subline SK-OV-3 MDR1-M6/6 ( Table 4). In these cell lines, compound 4 was again the most potent compound in the series. Comparison of the IC 50 values in the parental SK-OV-3 and genetically manipulated SK-OV-3 MDR1-M6/6 cell line allows for the calculation of a relative resistance value, designated Rr. This value is calculated by dividing the IC 50 value obtained in the Pgpexpressing SK-OV-3 MDR1-M6/6 cells by the IC 50 obtained in the parental SK-OV-3 cells. Paclitaxel, a known Pgp substrate, has an Rr value of 240, while CA-4, a poor Pgp substrate, has an Rr value of 1.3 (Table 4). Compound 4 had IC 50 values in SK-OV-3 and SK-OV-3 MDR1-M6/6 cells comparable to that of CA-4 and an Rr of 1.5, indicating that it is able to overcome drug resistance mediated by Pgp. Here, a correlation between a cell-based assay and a biochemical assay is not always observed, which might be due in part to the ability of the compounds to cross the cell membrane and accumulate intracellularly. Compounds 5-8, 10, 12, and 13 also had Rr values ≤ 1.5, suggesting that they are all poor substrates for Pgp-mediated transport and have advantages over the taxanes and vinca alkaloids in multidrug-resistant cancer cells.

Activity of Compound 4 in the NCI Cancer Cell Line Panel
Compound 4, the most potent compound of the series, was selected for evaluation in the NCI-60 cancer cell line panel [50], and it had a GI 50 (concentration causing 50% inhibition of cell proliferation) of~10 nM against 40 of the 60 cancer cell lines (Table 5). Compound 4 had better potency than the lead compound 1 in 50 cancer cell lines. (better by 5 to 6-fold in leukemia, 2 to 17-fold in NSCLC, 2 to 6-fold in colon cancer, 2 to 5-fold in CNS cancer, 2 to 25-fold in melanoma, 2 to 5-fold in ovarian cancer, 2 to 9-fold in renal cancer, 2 to 5-fold in prostate cancer, and 2 to 6-fold in breast cancer compared to lead compound 1) [26]. Thus 4, the thiophene-fused analogue, is up to 25-fold more potent than our previously published lead [26]. Compound 4 was selected for further evaluation in an in vivo xenograft mouse study in light of its nanomolar potency in vitro in the NCI-60 cancer cell line panel and its potent microtubule depolymerization activity. The in vivo effects of 4 were tested in the MDA-MB-435 xenograft model ( Figure 5). After conducting initial dose tolerance testing, 4 was administered at a dose of 75 mg/kg 3 × a week where it caused moderate weight loss yet had statistically significant antitumor effects as compared to the control at day 14, the end of the trial. In this trial, there was a trend toward antitumor effects with paclitaxel (15 mg/kg), but this did not reach statistical significance at any day or at trial conclusion.
in light of its nanomolar potency in vitro in the NCI-60 cancer cell line panel and its potent microtubule depolymerization activity. The in vivo effects of 4 were tested in the MDA-MB-435 xenograft model ( Figure 5). After conducting initial dose tolerance testing, 4 was administered at a dose of 75 mg/kg 3 × a week where it caused moderate weight loss yet had statistically significant antitumor effects as compared to the control at day 14, the end of the trial. In this trial, there was a trend toward antitumor effects with paclitaxel (15 mg/kg), but this did not reach statistical significance at any day or at trial conclusion.

Chemistry
All evaporations were carried out under a vacuum using a rotary evaporator. Analytical samples were dried in vacuo (0.2 mmHg) in a CHEM-DRY drying apparatus over P2O5 at 50 °C. Thin-layer chromatography (TLC) was performed on Whatman Sil G/UV254 silica gel plates (Whatman International Ltd., Maidstone, England), and the spots were

Chemistry
All evaporations were carried out under a vacuum using a rotary evaporator. Analytical samples were dried in vacuo (0.2 mmHg) in a CHEM-DRY drying apparatus over P 2 O 5 at 50 • C. Thin-layer chromatography (TLC) was performed on Whatman Sil G/UV254 silica gel plates (Whatman International Ltd., Maidstone, England), and the spots were visualized by irradiation at 254 nm. Proportions of solvents used for TLC are by volume. All analytical samples were homogeneous on TLC in at least two different solvent systems. Column chromatography was performed on a 70-230 mesh silica gel (Fisher Scientific, Waltham, MA, USA) column. The amount (weight) of silica gel for column chromatography was in the range of 50-100 times the amount (weight) of the crude compounds being separated. Columns were wet-packed with appropriate solvent unless specified otherwise. Melting points were determined using a digital MEL-TEMP II melting point apparatus with FLUKE 51 K/J electronic thermometer or using an MPA100 OptiMelt (Stanford Research Systems, Sunnyvale, CA, USA) automated melting point system and are uncorrected. Nuclear magnetic resonance spectra for protons ( 1 H NMR) were recorded on Bruker Avance II 400 (Billerica, MA, USA) (400 MHz) and 500 (500 MHz) systems and were analyzed using MestReC NMR (Mestrelab research, San Diego, CA, USA, data processing software. The chemical shift (δ) values are expressed in ppm (parts per million) relative to tetramethylsilane as an internal standard: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad singlet; exch, protons exchangeable by addition of D 2 O.
Elemental analyses or high-performance liquid chromatography (HPLC)/mass analysis were used to determine the purities of the target compounds. Elemental analyses were performed by Atlantic Microlab, Inc., Norcross, GA, USA. Elemental compositions are within ±0.4% of the calculated values and indicate >95% purity. Fractional moles of water or organic solvents found in some analytical samples could not be removed despite 24-48 h of drying in vacuo and were confirmed where possible by their presence in the 1 H NMR spectra. Mass spectral data were acquired on an Agilent G6220AA TOF LC/MS system using the nano ESI (Agilent chip tube system with infusion chip). HPLC analysis was performed on a Waters HPLC system using a XSelect CSH C18 column. Peak area of the major peak versus other peaks was used to determine purity. All solvents and chemicals were purchased from Sigma-Aldrich Co, USA. or Fisher Scientific Inc, USA. and were used as received.  (25 mL). The mixture was stirred at room temperature for 1 h, then at 60 • C for 12 h. The reaction mixture was cooled to room temperature, and the solvent was removed in vacuo. The crude product was purified by flash column chromatography on a silica column using hexane/ethyl acetate (10:1) as eluent to obtain compound 16 (7. The POCl 3 was evaporated, and the mixture was cooled in an ice bath. The mixture was neutralized using an aqueous NH 4 OH solution to yield a precipitate. The precipitate was collected by filtration, washed with water, dried and dissolved in MeOH. To the solution was added silica gel (1 g), and the solvent was removed under reduced pressure to provide a silica gel plug. Column chromatography was performed with hexane and ethyl acetate (10:1) to generate 20 (2.0 g, 6.19 mmol, 70%) as a brown solid. TLC Rf = 0.68 (hexane: EtOAc, 3:1); mp 234 • C; 1 H NMR (400 MHz, DMSO-d 6 ): δ 5.28 (s, br, 2H, exch., 2-NH 2 ), 2.74 (t, 2H, -CH 2 ), 1.92 (t, 2H, -CH 2 ), 1.56-1.50 (m, 2H, -CH 2 ), 1.43-1.38 (m, 2H, -CH 2 ). This compound was used for the next reaction without further characterization. 4-Chloro-5,6,7,8-tetrahydrobenzo [4,5]thieno [2,3-d]pyrimidine (21): Treatment of 16 (5.0 g, 22.19 mmol) with formamide (4.42 mL, 110.96 mmol) was carried out in a microwave vessel at 180 • C for 12 h. The reaction was cooled to room temperature, and 50 mL water was added to the mixture. The precipitate was collected and dried under high vacuum to afford 18 as a white solid in 72% yield (3.30 g). The product 18 was taken to the next step without characterization. Chlorination of 18 (3.0 g, 14.54 mmol) was performed using POCl 3 (1.4 mL, 14.54 mmol) and pyridine (1.17 mL, 14.54 mmol), and the mixture was kept at reflux for 8 h. The solvent was removed by evaporation, and the residue was neutralized with ammonia in water solution to generate a pale-yellow precipitate. The precipitate was collected by filtration. To the precipitate was added methanol and 2.0 g of silica gel. The solvent was removed under reduced pressure, and a silica plug was prepared. A flash column chromatographic separation was performed using ethyl acetate-hexane as eluent to afford 1.96 g of 21 ( Compounds 20-22 were dissolved in isopropanol, followed by addition of 1-2 drops of HCl and the appropriate anilines. The reaction mixture was stirred for 4-8 h at reflux. The reaction mixture was cooled, and silica gel was added to the solvent mixture to prepare a silica gel plug. A flash column chromatographic separation was performed using ethyl acetate-hexane as eluent to afford 4-7, 9, 11, 12 and 14 with yields of 48-68%. Compounds 9, 11 and 14 were added to NaH in DMF with drop-wise addition of iodomethane to obtain 8, 10 and 13, respectively, in 57-70% yield. N4-(4-methoxyphenyl)-N4-methyl-5,6,7,8-tetrahydrobenzo [4,5]thieno [2,3-d]pyrimidine-2,4-diamine (4): To a solution of 20 (250 mg, 1.04 mmol) in isopropanol (20 mL), 1-2 drops of HCl were added, followed by addition of 4-methoxy-N-methylaniline (157.3 mg, 1.15 mmol), followed by reflux for 6 h. The reaction mixture was cooled to room temperature, silica gel (500 mg) was added, and the solvent was removed under reduced pressure. Purification was performed by column chromatography using 1% MeOH in CHCl 3 as the eluant, and fractions containing the product (TLC) were pooled. The solvent was evaporated to give a white solid that was washed with CHCl 3 to afford 230. N4-methyl-N4-(4-(methylthio)phenyl)-5,6,7,8-tetrahydrobenzo [4,5]thieno [2,3-d] pyrimidine-2,4-diamine (5): To a solution of 20 (150 mg, 0.625 mmol) in toluene (8 mL), 1-2 drops of HCl were added, followed by addition of N-methyl-4-(methylthio)aniline (105.5 mg, 0.688 mmol), and the mixture was kept under reflux for 6 h. The reaction mixture was cooled to room temperature, silica gel (500 mg) was added, and the solvent was removed under reduced pressure. Purification was performed by column chromatography using 1% MeOH in CHCl 3 as the eluant, and the fractions containing the product (TLC) were pooled. The solvent was evaporated to give a pale-yellow solid that was washed with CHCl 3 to afford 109.

Molecular Modeling
Docking of target compounds 4-14 was carried out in the colchicine site of tubulin (PDB: 6BS2, 2.65 Å). The crystal structure PDBs were obtained from the protein database. All docking procedures were performed using various modules of the Schrödinger Maestro suite (Schrödinger, LLC, New York, NY, USA, 2020-2) [49]. The protein was optimized and prepared for docking using the Maestro Protein Preparation Wizard to assess bond order and add missing hydrogens, followed by energy minimization using the OPLS3e force field. Gaps in the protein structures were ignored, as they were far from the active site. The Maestro Induced-fit Grid Generation module was then used to define a 15 × 15 × 15 Å grid from the center of all the ligands. Ligands used in the computational docking study were built using the Maestro 2D Build module. The Maestro LigPrep module was then used to generate conformers of each compound subjected to energy minimization using the OPLS3e force field protocol. The resulting compounds were docked into the prepared protein using the Maestro Induced Fit Docking. Induced Fit Docking was performed with standard precision with flexible ligand sampling. A total of 20 initial poses were generated for each compound. Based on the pose score, the top 4 poses were selected and subjected to energy minimization using the OPLS3e force field. Finally, the top 2 poses per compound were generated and ranked according to the Glide score, which is an approximation of binding energy defined by receptor-ligand complex energies. The top pose was analyzed and presented in the Biological Evaluation and Discussion section. Docking scores are listed in Table S1 (Supplementary Materials).

Effects of Compounds on Cellular Microtubules
The effects of the compounds on cellular microtubules were evaluated in A-10 cells using indirect immunofluorescence microscopy. These cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were treated with the compounds of interest for 18 h, and the cells fixed with cold MeOH and microtubule structures were visualized using a β-tubulin antibody (Sigma-Aldrich, St. Louis, MO, USA). The concentration that caused loss of 50% of the interphase microtubules was defined as the EC 50 and calculated as previously described [52]. These values represent an average of at least three independent experiments.

Sulforhodamine B (SRB) Assay
The antiproliferative and cytotoxic effects of the compounds in cancer cells were evaluated using the SRB assay [53] as previously described [54]. MDA-MB-435 cells were obtained from the Lombardi Cancer Center of Georgetown University (Washington, DC, USA). SK-OV-3 and HeLa cells were purchased from ATCC. Details about the generation of the SK-OV-3 MDR1-M6/6 and HeLa WTβIII cells were described previously [54]. The IC 50 values represent an average of three independent experiments, each conducted using triplicate points.

Quantitative Tubulin Studies
Bovine brain tubulin was purified as described previously [55]. The tubulin assembly assay has been described in detail [50]. Briefly, 1.0 mg/mL of tubulin (10 µM) was preincubated for 15 min at 30 • C in 0.8 M monosodium glutamate (pH of 2 M stock solution adjusted to 6.6 with HCl), varying compound concentrations and 4% (v/v) DMSO as compound solvent. After the preincubation, the reaction mixtures were placed on ice and